The overall goal of this project is to establish new and novel methods and materials that will improve the low temperature storage of mammalian embryonic stem cells (ESCs). Here we propose to work only with mouse (i.e. non-human) ESCs. This work will result in intellectual property for the production of devices and disposables (e.g., media). ESCs are important biomedical research resources due to their broad applications in the areas of comparative medicine, comparative genomics, pharmacology, embryogenesis, and regenerative medicine. To fully utilize the potential of ESCs, it is necessary to have reliable methods for ESC maintenance, transportation, and cryopreservation. However, current cryopreservation protocols for ESCs remain suboptimal and highly variable. In order to improve cryopreservation methods for ESCs of all species, it is essential to understand their fundamental cryobiology in order to efficiently predict optimized protocols that are easily incorporated into routine laboratory use for both small and large scale ESC preservation. We will conduct a series of experiments to address these issues currently restricting optimal utilization of ESC and develop commercializable methods and products (devices and disposables) to implement these new, optimal cryopreservation methods. PUBLIC HEALTH RELEVANCE: Embryonic stem cells (ESCs) are important because of their broad potential applications in human medicine. However, current cryopreservation methods for ESCs are suboptimal and highly variable. The goal of this project is to establish novel methods and devices that improve cryopreservation of ESCs.